Single-stranded RNA viruses infecting the invasive Argentine ant, Linepithema humile

Abstract

Social insects host a diversity of viruses. We examined New Zealand populations of the globally widely distributed invasive Argentine ant (Linepithema humile) for RNA viruses. We used metatranscriptomic analysis, which identified six potential novel viruses in the Dicistroviridae family. Of these, three contigs were confirmed by Sanger sequencing as Linepithema humile virus-1 (LHUV-1), a novel strain of Kashmir bee virus (KBV) and Black queen cell virus (BQCV), while the others were chimeric or misassembled sequences. We extended the known sequence of LHUV-1 to confirm its placement in the Dicistroviridae and categorised its relationship to closest relatives, which were all viruses infecting Hymenoptera. We examined further for known viruses by mapping our metatranscriptomic sequences to all viral genomes, and confirmed KBV, BQCV, LHUV-1 and Deformed wing virus (DWV) presence using qRT-PCR. Viral replication was confirmed for DWV, KBV and LHUV-1. Viral titers in ants were higher in the presence of honey bee hives. Argentine ants appear to host a range of' honey bee' pathogens in addition to a virus currently described only from this invasive ant. The role of these viruses in the population dynamics of the ant remain to be determined, but offer potential targets for biocontrol approaches.

Figure 1

Sampling sites in New Zealand. Both the samples for the initial metatranscriptomic screen (including Sanger sequencing; n = 2) for viruses and the additional samples taken for virus confirmation and quantification (RT-PCR; n = 3) are shown. Samples from the northern-most sites included Argentine ants that were observed raiding honey bee hives, while the other sites were urban and distant from beehives. The concentration of viruses is derived from a standard curve, normalised to the concentration of the internal reference gene Ndufa8 (and due to the normalisation is unit-less). The map was generated in R v 3.1.1 with the packages ‘maps’, ‘mapdata’,‘maptools’ and ‘GISTools’. Full details of sites sampled are presented in Supplementary Table S3).

Figure 2

MEGAN taxonomic assignment of metatranscriptomic contigs. Putative viral sequences were based on BLAST similarity obtained using virus blastx matches for samples of Argentine ants from northern and southern samples. The MEGAN results were further refined using Virusfinder, Bowtie 2 and RT-PCR (Table 1).

Figure 3

Organization of Linepithema humile virus-1 LHUV-1 partial genome. The total nucleotide sequence length that was recovered from our analysis is indicated on the left of the figure. The light grey box indicates the predicted open reading frame (ORF1) of 1930 amino acids. Darker grey boxes identify protein motifs and their position within the ORF. The jagged grey box indicates the partially recovered ORF2, which contained a sequence that matched Dicistroviridae capsid proteins. Identifiable conserved ssRNA virus protein domains in ORF1 (Hel = helicase [position 549–664], Pro = Protease [position 1153–1381], RdRp = RNA-dependent RNA polymerase [position 1433–1927]) are indicated.

Figure 4

Phylogenetic tree of the Dicistroviridae ORF1 region. The tree includes the extended contig n1905 (proposed KBV strain) and LHUV-1 using a Le & Gascuel (LG) model with 500 bootstrap replicates. The ORF1 sequences included in the analysis comprise the complete Helicase, Protease and Ribosomal dependent RNA polymerase (RdRp) domains of these viruses and intergenic regions. Macrobrachium rosenbergii Taihu virus and Mud crab discistrovirus-1 (two unclassified Dicistroviridae that had ~50% identity to our contigs in blastp results) were included to root the tree. Our sequences are shown in bold text. GenBank accession numbers are shown in brackets.

Figure 5

Phylogenetic tree of viruses in ants and other Hymenoptera. The tree (a) was inferred using complete Ribosomal dependent RNA polymerase (RdRp) sequences, based on a Le & Gascuel (LG) model with Gamma parameter and invariant sites (LG+G+I) with 500 bootstrap replicates. A TSA that is a putative virus of the ant Monomorium pharoanis was also included. Our sequences are shown in bold text. Taxa found in ants are underlined. GenBank accession numbers are shown in brackets. The inset figure (b) shows the Aparavirus genus of Dicistroviridae, with bootstrap support. Solenopsis invicta virus 1,2 and 3 (SINV-1, SINV-2, SINV-3); Formica exsecta virus 1 (FEX-1); Deformed wing virus (DWV); Kashmir bee virus (KBV); Acute bee paralysis virus (ABPV); Black queen cell virus (BQCV); Chronic bee paralysis virus (CBPV); Israeli acute paralysis virus (IAPV); Monomorium pharaonis TSA (Mpha TSA); Varroa destructor virus 1 (VDV-1); Nylanderia fulva virus 1 (NfV-1); Linepithema humile virus 1 (LHUV-1); Nasonia vitripennis virus 1 (Nvit-1); Nasonia vitripennis virus 2 (Nvit-2).